KR20070053762A - Lipase inhibitor - Google Patents

Abstract

The present invention provides a lipase inhibitor containing gallotanine, an ellagitannin component fractionated from tea or Tellima grandiflora, and a food beverage and a pharmaceutical product to which the inhibitor is added.

The present invention, the following formula I:

(Wherein R ₁ , R ₂ and R ₃ are each independently H or gallic acid residues, R ₄ and R ₅ are H or gallic acid residues, or R ₄ and R ₅ become one and are of the formula:

Form the HHDP group, but

Provided that R ₁ to R ₅ At least two of which are gallic acid residues or when R ₁ , R ₂ and R ₃ are all H, R ₄ and R ₅ become one to form HHDP group)

Provided are lipase inhibitors containing at least one kind of compound represented by the above, and food and beverage and pharmaceuticals to which the inhibitors are added.

Lipase, gallotanine, ellagitannin

Description

Lipase inhibitors {LIPASE INHIBITOR}

This invention provides the lipase activity inhibitor which consists of a galotenin and an ellagitannin component which were fractionated from the tea leaf and Tellima grandiflora.

In recent years, ingestion of the high-fat diet has been on the rise as the Japanese lifestyle has become more and more. According to the 1999 National Nutrition Survey, although the energy intake decreases year by year, the ratio of lipid energy exceeds 25%, which is an appropriate ratio, and the proportion of people with high triglyceride or cholesterol levels is 5-6 halo when they are 60 years old or older. There is a report that has been confirmed (Summary of Ministry of Health, Labor and Welfare 1999 National Nutrition Survey, Clinical Nutrition 2001;

Obesity is one of the most serious diseases in modern society, but its main factor is the excessive intake of fat. In addition, excessive intake of fat is known to cause diabetes, hyperlipidemia, hypertension, atherosclerosis, and the like caused by obesity. In Japan, as a treatment for obesity, marginal stones (registered trademark) of an appetite suppressant are approved only in Japan, but side effects such as dry mouth, constipation, stomach discomfort, nausea and vomiting have been reported (clinical evaluation 1985; 13). (2): 419-459, clinical evaluation 1985; 13 (2): 461-515). Overseas, Zenical (registered trademark), which has a lipase inhibitory activity and inhibits fat absorption from the intestine, is commercially available as an obesity improving drug, but also fatty stool, an increase in the number of bowel movements, stool, and diarrhea. And side effects such as abdominal pain have been reported and are not necessarily safe (Lancet 1998; 352: 67-172).

In order to prevent obesity, reducing calorie intake by dietary restriction is an effective means, but practice in daily life is often difficult because it requires strict nutritional guidance. Therefore, it is thought that safe and healthy suppression of the absorption of diet-derived fat into the body is a practical and useful measure for the purpose of treating obesity and related diseases or improving health.

Under this background, the development of certain health foods that are safe and effective for the human body has been attracting attention. To date, food materials that suppress the rise of serum triglyceride levels after meals include globin protein degradation products that inhibit fat absorption by inhibiting pancreatic lipase (J. Nutr. 1988; 128: 56-60, Japanese Clinical and Food Journal 1999; 52 (2): 71-77, Health and Nutrition Food Research 2002; 5 (3): 131-144), diacylglycerols having different digestive absorption properties than triacylglycerols (J. Am. Coll. Nutr. 2000 19 (6): 789-796, Clin. Chim. Acta. 2001; 11 (2): 109-117), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc. purified from fish oil; It is released as this specific health food.

Plant-derived lipase inhibitory active substances have also attracted attention in recent years. In particular, regarding polyphenols having lipase-inhibiting activity, tannins derived from plant bark (Japanese Patent Publication No. 60-11912), tannins contained in legumes, Flavonoids and its glycosides (Japanese Patent Application Laid-Open No. Hei 8-259557), lipid absorption inhibitory foods containing the main components Epigarocatechin garate and epicatechin garate in green tea (Japanese Patent Laid-Open Application No. Hei 3-228664), bell pepper and shimeji Lipase inhibitors (Japanese Patent Laid-Open Publication No. 3-219872), flavones and flavonols (Japanese Laid-Open Patent Publication Hei 7-61927) consisting of water extracts such as mushrooms, pumpkins, leafy mushrooms, green radish, green tea and oolong tea Benzoic acid (gallic acid) (JP-A-11-102022), triterpene compounds and derivatives thereof (JP-A-9-40689) and tamarind procyanidins Anti-obesity agents (Japanese Patent Application Laid-open No. Hei 9-291039) and the like have been reported, and lipase inhibitory action of grape seed extract (Nutrition 2003; 19 (10): 876-879), caused by polysaccharide derived from sarasia Lipase inhibitory activity and anti-obesity effect of rats (J. Nutr. 2002; 132: 1819-1824), and anti-obesity action of mice by oolong tea extract (Int. J. Obes. 1999; 23: 98-105) Known.

However, the lipase inhibitor derived from the botanicals shown above cannot be said to have sufficient effect. For example, even if it is effective in extracts of certain plants, since natural products are of origin unless the amount of the active ingredient contained therein is clear, it is difficult to stably maintain lipase inhibitory activity. Moreover, in the case of an inhibitor derived from a plant with low palatability, there exists a problem that it affects a flavor to use as food. For example, a report showing the lipid-improving effect of oolong tea reported that a significant decrease in blood triglyceride levels was confirmed by drinking commercial oolong tea 1330 ml / day for 6 weeks (Japanese Nutrition and Food Journal 1991; 44 ( 4): 251-259) B. In contrast to 102 men and women with simple obesity, oolong tea (2g × 4 / day) was taken orally for 6 consecutive weeks, and 67% of subjects found a weight loss of 1 kg or more. In addition, there are reports of significant improvement after oolong tea intake in subjects with high blood triglyceride levels (Japanese Clinical Nutrition Journal 1998; 20 (1): 83-90). Although the effects have been confirmed in the large-scale drinking of oolong tea, it is difficult to continue in daily life. Moreover, even if it provides simply concentrated oolong tea, since bitterness and astringent taste are strong and the amount of caffeine increases, it is not suitable as a realistic measure.

On the other hand, tea-derived tannins have been reported for their antioxidant activity (Biosci. Biotechnol. Biochem. 2003; 67 (2): 396-401), and plant-derived tannins, especially tellimagrandin, have antioxidant activity (Phytochemistry 1993; 33 (3): 557-561) as well as antimicrobial (Microbiol. Immunol. 2004 ,; 48 (1): 67-73) anti-cancer (Toxicology Letters 2004; 147 (2): 109-119), cytokine release modulators (Japan (Patent Publication 2004-510688) and the like have been reported.

Patent Document 1: Japanese Patent Publication No. 60-11912

Patent document 2: Unexamined-Japanese-Patent No. 8-259557

Patent document 3: Unexamined-Japanese-Patent No. 3-228664

Patent document 4: Unexamined-Japanese-Patent No. 3-219872

Patent document 5: Unexamined-Japanese-Patent No. 7-61927

Patent Document 6: Japanese Patent Application Laid-Open No. 1-02022

Patent Document 7: Japanese Patent Application Laid-Open No. 9-40689

Patent Document 8: Japanese Patent Application Laid-Open No. 9-291039

Patent Document 9: Japanese Patent Application Publication 2004-510688

[Non-Patent Document 1] Summary of 11-year National Nutrition Survey, Ministry of Health, Labor and Welfare

Non Patent Literature 2: Clinical Nutrition 2001; 98 (5): 577-588

Non Patent Literature 3: Clinical Evaluation 1985; 13 (2): 419-459

Non Patent Literature 4: Clinical Evaluation 1985; 13 (2): 461-515

Non Patent Literature 5: Lancet 1998; 352: 67-172

[Non-Patent Document 6] J. Nutr. 1988; 128: 56-60

Non-Patent Document 7: Japanese Society for Clinical and Food Science 1999; 52 (2): 71-77

Non Patent Literature 8: Health and Nutrition Food Research 2002; 5 (3): 131-144

Non-Patent Document 9: J. Am. Coll. Nutr. 2000; 19 (6): 789-796

Non Patent Literature 10: Clin. Chim. Acta. 2001; 11 (2): 109-117

Non Patent Literature 11: Nutrition 2003; 19 (10): 876-879

Non Patent Literature 12: J. Nutr. 2002; 132: 1819-1824

Non Patent Literature l3: Int. J. Obes. 1999; 23: 98-105

Non-Patent Document 14: Japanese Society for Nutrition and Food, 1991; 44 (4): 251-259

Non Patent Literature 15: Japanese Society of Clinical Nutrition 1998; 20 (1): 83-90

Non Patent Literature 16: Biosci. Biotechnol. Biochem. 2003; 67 (2): 396-401

Non Patent Literature 17: Phytochemistry 1993; 33 (3): 557-561

Non Patent Literature 18: Microbiol. Immunol. 2004; 48 (1): 67-73

Non Patent Literature 19: Toxicology Letters 2004; 147 (2): 109-119

Disclosure of the Invention

Problems to be Solved by the Invention

The present invention focuses on various polyphenol components contained in plants, and provides lipase inhibitors containing at least one of gallotanine and ellagitannin components fractionated from tea leaves and Tellima grandiflora.

In addition, the present invention provides a food and beverage for the purpose of reducing the triglycerides in the blood and improving health without adding the lipase inhibitor to inhibit flavor.

The present invention also provides a pharmaceutical composition containing the lipase inhibitor and suppressing the absorption of fat derived from a meal to suppress the rise of triglycerides in the blood.

Means to solve the problem

As a means to solve the above problems, a compound having a plurality of gallic acid in the molecule by finding a component that inhibits pancreatic lipase essential for fat absorption from tea and Tellima grandiflora, evaluating the lipase inhibitory activity of various polyphenols present therein It was found that there is a strong lipase inhibitory activity.

More specifically, the lipase inhibitor of the present invention is represented by the following formula I:

(Wherein R ₁ , R ₂ and R ₃ are each independently H or gallic acid residues, R ₄ and R ₅ are H or gallic acid residues, or R ₄ and R ₅ become one and are of the formula:

Form the HHDP group, but

Provided that at least two of R ₁ to R ₅ are gallic acid residues, or when R ₁ , R ₂ and R ₃ are all H, R ₄ and R ₅ become one to form an HHDP group)

At least one kind of compound represented by this is made into an active ingredient. In addition, in this specification, a gallic acid residue means the residue of the gallic acid in which OH was taken from the carboxyl group.

Preferred active ingredients are compounds in which at least three of R ₁ to R ₅ are not H in the formula (I).

A more preferable active ingredient is a compound in formula I in which at least 4 of R ₁ to R ₅ are not H.

One more preferred active ingredient is a compound wherein, in Formula I, all of R ₁ to R ₅ are not H, for example, all of R ₁ , R ₂ and R ₃ are gallic acid residues, and R ₄ and R ₅ are one To form an HHDP group (compound 8).

As an example of the specific compound contained in the lipase inhibitor of this invention, the compound shown in FIG. 1 is mentioned.

Lipase inhibitors

The lipase inhibitory active compound of the present invention may be used alone as a lipase inhibitor without containing other components, or may be used as a lipase inhibitor with a solvent or a solid carrier. It is preferable that a solvent or a carrier can be used safely as a food or a medicine in consideration of the use as the following food and beverage and / or a medicine. The lipase inhibitors of the present invention have a variety of uses, for example, for use in test studies, as an active ingredient in foods and pharmaceuticals to prevent the accumulation of triglycerides.

Method for measuring lipase inhibitory activity

Lipase inhibitors of the present invention have a strong inhibitory action against lipases, in particular pancreatic lipases. The inhibitory activity can be measured by the method specifically described in Example 1.

Food and drink containing lipase inhibitor

The lipase inhibitor of the present invention can be added to food and drink to prevent undesired rise of triglycerides in the blood accompanying ingestion of fat from meals and / or reduce elevated triglycerides in the blood. Preferred examples of the food and drink are food and drink ingested daily, for example, green tea, barley tea, oolong tea, black tea, coffee, sports drinks, beverages, seasonings, dressings. But food and drink should just eat normally, soft drink, cocktail, beer, whiskey, shochu, wine, sake, seasoning, dressing, seasoning, processed food, convenience food, retort food, chocolate, fresh cream, confectionery, dairy product, healthy food Or a supplement may be used.

The amount of the lipase inhibitor of the present invention added to the food and drink is added so that the intake amount of the active ingredient per meal is 0.1 mg to 10 g. However, since the lipase inhibitor of the present invention is derived from food, the safety is very high, and there is no substantial upper limit to the amount of the lipase added to the food and beverage.

Drugs containing lipase inhibitors

The lipase inhibitor of the present invention can also be used as an active ingredient of a medicament for inhibiting absorption of fats derived from meals and preventing and / or lowering undesirable rise of triglycerides in the blood. Preferred medicaments are medicaments administered orally, and examples thereof include drinks, tablets, capsules, granules, powders, candy, and drops. The amount of the lipase inhibitor of the present invention contained in the drug is 0.1 mg to 10 g per dose.

Since the drug of the present invention has high safety of the lipase inhibitory component, it is safe even if taken over a long period of time. Therefore, in order to prevent or eliminate obesity as a lifestyle disease, it is also possible to take it routinely.

Effects of the Invention

The present invention adds a lipase inhibitor containing at least one of gallotanine and ellagitannin components derived from tea leaves and Tellima grandiflora, and does not inhibit flavor, has high palatability, and aims to reduce triglycerides and improve health. One can provide food and drink. In order to suppress absorption of dietary fat, it is preferable to take it with a meal, and the drink which strengthened the active ingredient obtained from tea is significant. In particular, by enhancing these components, it is possible to provide a beverage for the purpose of anti-obesity action and health promotion.

Brief description of the drawings

1 shows chemical structural formulas of compounds used for lipase inhibitory activity evaluation in Example 5. FIG.

Example 1 Measurement of Lipase Inhibitory Activity

The measurement of the lipase activity was performed by measuring the fluorescence of 4-methyl unberiferone produced by the reaction using fluorescent oleic acid ester (4-UMO) of 4-methyl unberiferon as a substrate.

In the measurement, 13 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 1.36 mM CaCl ₂ was used as the buffer. 4-UMO (manufactured by Sigma), which is a substrate, was prepared as 0.1 M DMSO solution by 1000 times dilution with the buffer, and lipase was 400 U using the same buffer as pig pancreatic lipase (manufactured by Sigma). What was prepared as / mL solution was used for the enzyme measurement.

The enzyme reaction was initiated by adding 50 μl of 4-UMO buffer solution and 25 μl of distilled water (or sample solution) to a 96 well microplate under 25 ° C., followed by addition of 25 μl of lipase buffer solution. . After the reaction was carried out for 30 minutes, the reaction was stopped by adding 100 µl of 0.1 M citric acid buffer (pH4.2), and the fluorescence (excitation wavelength 355 nm, fluorescence wavelength 460 nm) of 4-methylunberiferone produced by the reaction was stopped. ) Was measured using a fluorescent plate reader (Fluoroskan Asent CF manufactured by Labsystems).

The inhibitory activity of the test sample was determined as a sample amount IC ₅₀ (μM) that inhibits 50% of the control (distilled water) activity.

Measure sample

Gallic acid (Compound 1) was purchased from Nacalai task. Compounds 2, 3, 4, and 5 were prepared by Hashimoto's paper (Chem. Pharm. Bull. 1989; 37 (12): 3255-3563, Chem. Pharm. Bull. 1989; 37 (1): 77-85). Purification from oolong tea (Example 2). 1,2,4,6-tetragalloylglucose (1,2,4,6-tetragalloylglucose) (Compound 6) was purified from Camellia ptilophylla by the method of Example 3. Tellimagrandin (Compound 7,8) was purified from Tellima grandiflora by the method of Example 4.

Example 2 Purification of Galotenins 2,3,4,5

Compounds 2, 3, and 5 were purified by the method of Hashimoto et al. (Chem. Pharm. Bull. 1989; 37 (12): 3255-3563). The method is simply shown below. The oolong tea leaves were extracted with 80% acetone, and then the acetone was removed and fractionated using water, methanol and 50% acetone with Sephadex LH-20 (manufactured by Palmacia). Water-methanol elution fractions were subjected to water-methanol elution with MCl-gel CHP-20P (manufactured by Mitsubishi Chemical Corporation) and loaded again on Sephadex LH-20 (manufactured by Palmacia) to obtain compounds 2 and 3 in the aqueous solution. . The water-methanol elution fraction was eluted with water-methanol using Sephadex LH-20 (manufactured by Palmasia), and then eluted with water-methanol using Fuji gel ODS-G3 (manufactured by Fuji-Silysia Chemical Co., Ltd.) to give compound 5 Got it.

Compound 4 is also described in the paper Chem. Pharm. Bull. 1989; 37 (1): It refine | purified as follows by the method of 77-85. The oolong tea leaves were extracted with 80% acetone, and then the acetone was removed and fractionated using water, methanol and 50% acetone with Sephadex LH-20 (manufactured by Palmacia). The methanol eluate was eluted with water-methanol with MCl-gel CHP-20P (manufactured by Mitsubishi Chemical Corporation), followed by water methanol eluting with Bondapak C18 (manufactured by Waters), and ethanol with Sephadex LH-20 (manufactured by Palmacia). Elution was carried out to obtain compound 4.

Example 3 Purification of 1,2,4,6-tetragalloylglucose (Compound 6)

100 g of leaves (dried) of Camellia ptilophylla were extracted for 4 minutes in 2000 ml of hot water (90 ° C.) and lyophilized to purify as follows. A 1% aqueous solution of the lyophilized powder was adsorbed onto a Sep-Pak C18 Cartridge (5 ml, manufactured by Waters). After washing with water, the fraction eluted with acetonitrile was lyophilized. This fraction, 250 mg, was loaded onto Develosil C30-UG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.), and a linear gradient of 5-30% acetonitrile in the presence of 0.05% TFA (5 ml / min, 180 min) Was eluted at and fractionated while monitoring the absorption at A280 nm. The obtained fractions were then loaded into YMC-Pak ODS (20 mm × 250 mm, manufactured by YMC Corporation) and eluted in a linear gradient of 20-25% acetonitrile (6 ml / min, 60 min) in the presence of 0.1% TFA. Purification was carried out to obtain 1,2,4,6-tetragalloylglucose (Compound 6).

Example 4 Tablets of Tellimagrandin Family

Phytochemistry 1976; 15: According to 211-214, extraction purification was performed as follows. 100 g of leaves of Tellima grandiflora were pulverized in liquid nitrogen, extracted with 1000 ml of 50% ethanol, the solvent was distilled off, loaded on adsorption resin HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.), washed with water, and washed with acetonitrile. The eluting fractions were lyophilized. This fraction was loaded onto Develosil ODS-UG-5 (50 mm x 500 mm, manufactured by Nomura Chemical Co., Ltd.) and eluted in a linear gradient of 5-25% acetonitrile (32 ml / min, 80 min) in the presence of 0.05% TFA. And fractionating while monitoring the absorption of A260 nm, Tellimagrandin 1 and Tellimagrandin 2 were obtained.

Example 5 Lipase Inhibitory Activity

Table 1 shows the lipase inhibitory activity of the tragacatechins and tellimagrandin (elagitannin) derived from tea. In order to examine the structural activity correlations, IC ₅₀ values were expressed in μM. In addition, the chemical structural formula of the compound used for evaluation was described in FIG.

Among these tannins, lipase inhibitory activity was shown to be a compound in which gallic acid was bound to sugar by at least 3 molecules (compounds 4,6) and ellagitannins (compounds 5,7,8) to which HHDP groups were bound. Glycosides with only one molecule of gallic acid or gallic acid were inactive. It has been found that the presence of at least two gallate groups in the molecule is required for active expression in that lipase inhibitory activity increases as more gallic acid increases.

Claims (9)

Expression I:

(Wherein R ₁ , R ₂ and R ₃ are each independently H or gallic acid residues, R ₄ and R ₅ are H or gallic acid residues, or R ₄ and R ₅ become one and are of the formula:

Form the HHDP group, but Provided that at least two of R ₁ to R ₅ are gallic acid residues, or when R ₁ , R ₂ and R ₃ are all H, R ₄ and R ₅ become one to form an HHDP group) Lipase inhibitor containing at least one kind of compound represented by. The method of claim 1, The lipase inhibitor of formula I, wherein at least three of R ₁ to R ₅ contain a compound other than H. The method of claim 1, A lipase inhibitor of formula I wherein at least 4 of R ₁ to R ₅ contain a compound other than H. The method of claim 1, A lipase inhibitor according to formula I, wherein all of R ₁ to R ₅ contain a compound other than H. The method of claim 1, The lipase inhibitor of Formula I containing the compound in which all of R <_1> , R <_2> and R <_3> are gallic acid residues, and R <_4> and R <_5> become one and form an HHDP group. Food and beverage which added the lipase inhibitor in any one of Claims 1-5. The method of claim 6, Food beverages selected from the group consisting of tea drinks, soft drinks and healthy food. The pharmaceutical composition containing the lipase inhibitor of any one of Claims 1-5. The method of claim 8, Pharmaceutical composition for suppressing absorption of fat derived from a meal.

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